Erythropoietin or "EPO" is a glycoprotein hormone produced in the kidney and the liver in response to hypoxia; it is a required growth factor for red blood cell production. The existence of a hormone that regulated erythropoiesis was first proposed at the beginning of this century by Carnot and deFlandre. However, because of difficulties in reproducing their results, the idea that a hormone was involved in the regulation of erythropoiesis fell into disfavor until 1943 when Krundieck was able to reproducibly demonstrate the presence of an erythropoietic substance in the serum of anemic rabbits. Subsequently, in 1950, Reissmann [Blood 5:372-380 (1950)] provided strong evidence for the humoral regulation of erythropoiesis using an elegant parabiotic rat model; and in1953 Erslev [Blood 8:349-357 (1953)] provided still more evidence that a hormone, erythropoietin, was involved in the regulation of red cell production. Erslev demonstrated that plasma taken from an anemic rabbit and injected into a non-anemic rabbit caused a dose-dependent reticulocytosis and increase in erythroid precursors in the bone marrow. Prior to birth, EPO is produced primarily in the liver; whereas after birth, the kidney becomes the major endogenous EPO producing organ.
Despite considerable effort, attempts at purification of EPO were elusive until 1977 when Goldwasser and his colleagues [Miyake et al., J. Biol. Chem. 252:5558 (1977)] were able to isolate approximately 10 mg of homogeneous human EPO and subsequently determined a partial amino acid sequence. This achievement subsequently led to the isolation of genomic and cDNA clones of human and mouse EPO.
The human EPO was cloned in 1985 by two independent groups [Jacobs et al., Nature 313:806-810 (1985); Lin et al., Proc. Natl. Acad. Sci. USA 82:7580-7584 (1985)]. The sequence is highly conserved between mouse and man providing an indication of its evolutionary importance. The human EPO gene encodes a protein of 193 amino acids. Following cleavage of a 27 amino acid N-terminal "leader" sequence, the mature protein has a calculated molecular weight of about 18,400. However, human EPO contains approximately 40% carbohydrate, giving it a molecular weight of approximately 30,400 daltons. Post-translational glycosylation is clearly required for in vivo function. In addition, human EPO contains two disulfide bonds, at least one of which appears to be essential for the function of the molecule. Recombinant human EPO or "r-HuEPO" has been mass produced in Chinese hamster ovary cell lines and is indistinguishable from the native molecule. In clinical trials, to date, no patients have developed antibodies to r-HuEPO, thereby showing that for all intents and purposes r-HuEPO is virtually identical to the native endogenous human EPO.
Hypoxia is the chief stimulus for in vivo production of EPO. In response to the sensing of hypoxia in the kidney and the liver, increased EPO gene transcription in vivo leads to increase EPO mRNA and an increased production and secretion of EPO protein. The hormone (EPO) travels to hematopoietic tissues where it binds to its receptor on erythroid progenitor cells and stimulates them to proliferate and differentiate into mature red blood cells. This results in an increase in the oxygen carrying capacity of the blood, alleviating the hypoxic stimulus, and providing a complete feedback loop for regulation of EPO gene expression and red blood cell production. In anemic patients, serum EPO levels have been shown to be inversely proportional to the hematocrit or hemoglobin concentration providing that renal function is normal. The level of EPO in the plasma correlates with the rate of production of new erythrocytes by the bone marrow. Failure to increase the amount of circulating EPO in response to hypoxic stress can lead to anemia.
Prior to the cloning of the EPO gene, measurement of endogenous EPO in plasma and serum relied primarily on in vivo and in vitro bioassays. The flaw of relying solely on bioassays has been clearly demonstrated by Sytkowski et al. who discovered a renal cell carcinoma cell line with erythropoietin-like activity but which was immunologically distinct from erythropoietin when assayed by a sensitive and specific radioimmunoassay. Although an accurate immunologic assay for erythropoietin was first developed in 1979, readily available assays have only recently come into being. Hence, up until the past few years, there continued to be many studies which relied solely on bioassays to assess erythropoietin serum concentrations in various clinical settings. However, more and more studies are now performed using immunoassays as the technique of choice. Most of these studies have employed immunoassays with polyclonal antisera and, most recently, recombinant standards. Using such assays, normal serum levels of EPO lie in the vicinity of 4-30 mU/ml; with different assays yielding slightly different, but overlapping, normal ranges. Moreover, serum EPO levels have been confirmed to be inversely proportional to the hematocrit or hemoglobin concentration, providing that the patient has normal renal function.
Of particular interest is the finding that when normal individuals are sequentially phlebotomized (up to one unlit of blood, twice per week for three weeks), they generally increase their circulating EPO levels only to a modest degree. This observation reveals an important aspect of the natural regulation of erythropoiesis. In response to progressive anemia, and hence increasing degrees of tissue hypoxia, the increase in endogenous EPO production is gradual and graded. The low levels of EPO which are always present appear to be sufficient to allow for a basal rate of erythropoiesis. Thus, relatively small losses of blood, such as a one unit blood bank donation, stimulate in vivo EPO production only to a small degree; and the red cell mass slowly returns to its steady state level with only a small change in the rate of erythropoiesis. It is only after a major blood loss that a markedly increased production of EPO and rate of erythropoiesis ensue.
Within the last few years, however, a new phenomenon has presented itself. Since in-vivo use of exogenous erythropoietin, and typically recombinant, human EPO became recognized as stimulating erythropoiesis; a surreptitious use of recombinant EPO has become a favored elicit drug by athletes, both amateur and professional. The use of such elicit drugs is not permitted by almost all athletic bodies, and in particular by the International Olympic Committee and its affiliates. Nevertheless, the surreptitious use of EPO is believed to be common because, although the effects of the drug are longlasting enduring for many weeks, EPO has a relatively short half-life in-vivo within the body, lasting only a few hours to a few days at most. In addition, because many coaches as well as athletes are medically informed today, the prevalent practice now is to discontinue the surreptitious use of EPO a calendar week or so before the upcoming sports event. Thus, direct chemical determinations for EPO in the blood appear normal on the day of the sport event, it being impossible to distinguish between endogenous EPO made in-vivo and recombinant EPO whose use was discontinued earlier in time.
Insofar as is presently known, however, there are no reported investigations or published procedures which even suggest that an effective method and protocol for detecting a surreptitious use of erythropoietin might be possible. Given the ever increasing authorized and clandestine use and administration of rEPO, the generation of a rapid and reliable methodology by which to determine whether a living subject has been using EPO would be generally recognized and accepted as a major advance and long desired development by persons working in this field.